Autism spectrum disorders (ASD) currently affect about 1 in 110 children in the US, and are associated with a high economic cost to both families and society. There are currently no consistent biological markers for Autism, and diagnosis is currently based upon analysis of behavioral traits and developmental history. The most beneficial treatment for ASD is behavioral therapy, which is most effective if administered early in life. We have previously identified and validated a set of protein biomarkers for autism risk in the plasma of women from a large cohort of mothers of children with autism and unaffected controls. The main goal of the proposed research is to develop a multiplex quantitative assay for these biomarkers to be able to predict the risk of having a child with autism, prior to pregnancy, especially in those mothers who have at least one child with autism and are at elevated risk of having a second child on the spectrum. This test can also be used post-natally to determine if a child is at risk based on the maternal autoantibody profile. To achieve this goal we propose the following three Specific Aims: Aim #1: Express LDH-A, LDH-B, Cypin, STIP1, CRMP1, and CRIMP2 proteins in both a mammalian and prokaryotic expression systems. To evaluate the immunoreactivity of the endogenous autoantibodies from mothers of children with autism and typically developing controls against these expressed proteins. Aim #2: Develop high throughput multiplexed assays for LDH, Cypin, STIP1 and CRMP1. Produce standard curve of autoantibody concentration vs. signal in buffer and plasma and evaluate assay accuracy, precision, and sensitivity. Aim #3: Validation studies with blinded serum sets using the newly developed multiplex assays will be used to correlate the concentration of each of these endogenous autoantibodies to the development of disease in samples taken from mothers during pregnancy that gave birth to a child with autism vs. mothers that give birth to typically developing child, and establish a threshold for a positive prognosis with 99% confidence. The project will result in a diagnostic product that will give women an opportunity to know if they are at risk of having a child with autism prior to conception, thereby allowing the possibility to take preventative steps and early intervention.